Watching proteins function with time-resolved x-ray crystallography

Abstract

Macromolecular crystallography was immensely successful in the last two decades. To a large degree this success resulted from use of powerful third generation synchrotron X-ray sources. An expansive database of more than 100,000 protein structures, of which many were determined at resolution better than 2 Å, is available today. With this achievement, the spotlight in structural biology is shifting from determination of static structures to elucidating dynamic aspects of protein function. A powerful tool for addressing these aspects is time-resolved crystallography, where a genuine biological function is triggered in the crystal with a goal of capturing molecules in action and determining protein kinetics and structures of intermediates (Schmidt et al., 2005a; Schmidt 2008; Neutze and Moffat, 2012; Šrajer 2014). In this approach, short and intense X-ray pulses are used to probe intermediates in real time and at room temperature, in an ongoing reaction that is initiated synchronously and rapidly in the crystal. Time-resolved macromolecular crystallography with 100 ps time resolution at synchrotron X-ray sources is in its mature phase today, particularly for studies of reversible, light-initiated reactions. The advent of the new free electron lasers for hard X-rays (XFELs; 5–20 keV), which provide exceptionally intense, femtosecond X-ray pulses, marks a new frontier for time-resolved crystallography. The exploration of ultra-fast events becomes possible in high-resolution structural detail, on sub-picosecond time scales (Tenboer et al., 2014; Barends et al., 2015; Pande et al., 2016). We review here state-of-the-art time-resolved crystallographic experiments both at synchrotrons and XFELs. We also outline challenges and further developments necessary to broaden the application of these methods to many important proteins and enzymes of biomedical relevance.

Molecular dynamics simulations of macromolecular crystals

“…These studies aim to catch tiny crystals passing through a micrometer‐sized detector, whether on a stage or diffusing free in liquid. The time limit in this latter case is set by the timing of the pump laser and X‐ray probe pulses …”

Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM

“…First successes came from cryo-electron crystallography [8][9][10][11][12] by diffusing small interactants into crystals, broadly analogous to time-resolved X-ray crystallography techniques 13 and limited to crystalline samples and small diffusible ligands. More recent attempts in the framework of cryo-EM and single-particle analysis have traced structural changes over time in processes that proceed over several seconds and even minutes.…”

Violation of centrosymmetry in time-resolved coherent x-ray diffraction from rovibrational states of diatomic molecules

“…We consider the x-ray pulse has an axially symmetric Gaussian profile propagating in the x direction with velocity c: (39) where E 0 is the peak electric field, σ yz and σ x are the respective Gaussian widths in the transverse and longitudinal directions, and κ 0 is the central wave number. Substituting Eq.…”

“…Coherent x-ray diffraction [28][29][30][31], in particular, has long been used to determine the microscopic structures of molecules, solids, and proteins because of itsångström resolution and deep penetration depth [32]. With the recent development of femtosecond (fs) x-ray sources, time-resolved coherent x-ray diffraction (using a pump-probe scheme) has been shown capable of directly imaging the transient structures along a reaction path [33][34][35][36][37][38][39][40][41][42], thereby providing deeper insight into the underlying reaction mechanisms. Theories have also been developed to simulate and interpret the timeresolved diffraction images in terms of target electronic or molecular motions [43][44][45][46][47][48][49].…”